Amphiregulin (AR) is a member of the epidermal growth factor receptor (EGFR) family of ligands. In cultured human keratinocytes, amphiregulin is the major autocrine growth factor. Additionally, the mitogenic effects of AR on keratinocytes can be enhanced by interaction with other proteins such as CD9 and are inhibited by heparin. Although the role of AR in normal adult epidermis is not completely understood, analysis of AR protein and mRNA levels has provided some insights into its function. AR is weakly expressed in keratinocytes in normal adult epidermis but is up-regulated in several hyperproliferative disorders such as psoriasis and in tumors. A possible causal link between elevated AR levels and psoriasis has been suggested by the recent finding that transgenic mice overexpressing human AR cDNA under the Kl4 keratin promoter display a psoriatic phenotype. This result stands in direct contrast to the effects of TGFalpha overexpression, suggesting distinct roles for these two ligands. Moreover, AR, but not TGFalpha, is dramatically induced (10-30 fold) in several wounding models. Amphiregulin is synthesized as a glycosylated membrane-anchored precursor (proAR). In polarized epithelial cells, we have examined the biosynthesis and processing of proAR and demonstrated complex sequential processing of proAR ectodomain to produce multiple cellular and soluble AR forms. A predominant 43 kD soluble AR form is novel, contains the N-terminal proregion and has a C-terminal extension. The biological significance of multiple AR species is not known. The goals of this grant proposal are to identify the cellular and soluble forms of AR produced in normal human keratinocytes and begin to address their biological function in this cell type. These studies will be the first to comprehensively examine proAR biosynthesis and processing in a normal primary epithelial cell type. The mechanism(s) involved in regulating proAR ectodomain cleavage in keratinocytes will also provide the basis for understanding the role of metalloproteases in this process and more generally in the process of membrane protein shedding. Finally, these studies should enhance our understanding of the role of the various AR forms expressed under different environmental conditions (eg, UV exposure) and in different skin disease states.